Enzymatic ligation is essential for the synthesis of long DNA. However, a number of ligated products exponentially decay as DNA synthesis proceeds randomly. The control of ligation randomness is important to suppress exponential decay and achieve efficient synthesis of long DNA. This study analyzes the randomness in sequential DNA ligations, termed qPCR-based STatistical Analysis of Randomness (qPCR-bSTAR), using a probability distribution of ligated DNA length. Exponential decay is suppressed in a polymer solution. Further, DNA ligation is found to be activated at optimal crowded conditions. The theoretical model of kinetic ligation suggests that intermolecular attraction due to molecular crowding is involved in the optimal balance of ligation speed and available ligase. Our findings indicate that crowding effects enhance the synthesis of long DNA with large amounts of genetic information retained.